Objective To elucidate the effect of Ge-132 on the growth of melanocytes. Mothods Melanocyes from epidermis were cultured and purified ;the second generation of the cell was used for study ;the cells were divided into two groups randomly,to group A, Ge-132 was added to the media at 0.04mg/L ;to group B ,common culturing method was used without Ge-132. After 5d, the cells were seperated by digestion for study by transmission electronic micro- scope. ResultsCompared to group B, the vacuioes of the cells were increased,mitochondria distended, endoplasmic reticulum dilated and the number of melanosome declined in the group A. Conclusion Ge-132 can inhibit the melanocyte＇s growth at a certain concentration and might be used for treating pigmented diseases.

Objective To construct a recombinant plasmid DNA containing herpes simplex virus type 1(HSV-1) glycoprotein D (gD) gene.Methods The HSV-1 gD gene was obtained by polymerase chain reaction (PCR) and inserted into TA cloning vector pGEM-T, then cloned into the eukaryotic expression vector pcDNA3.1 to generate pLy-D. The recombinant plasmid pLy-D, which was confirmed by partial sequencing and restriction endonuclease analysis, was transfected into Cos-7 cells and used to inoculate ICR mice via muscular injection. Immunohistochemistry and enzyme-linked immunoabsorbent assay (ELISA) were employed to test the gD expression in transfected cells and the specific anti-HSV-1 antibody in the serum of immunized mice, respectively.Results The gD eukaryotic expression plasmid pLy-D was constructed. Using the immunohistochemistry technique, the gD expression in pLy-D-transfected cells was detected. The ELISA demonstrated that specific anti-HSV-1 antibody could be induced in immunized mice after three times injection.Conclusions We constructed HSV-1 gD eukaryotic expression plasmid pLy-D which could express gD protein in transfected cells and could induce humoral immune response in mice. This observation will be helpful in designing HSV prophylactic vaccine.

Objective To study the application of P1 adhesin protein epitopes in diagnosis of Mycoplasma pneumoniae(Mp) infected patient. Methods The major epitope(P1-534) of P1 adhesin protein were predicted by ProPred and ANTIGENIC according to its primary structure. The high value fragment was cloned into a constructed recombinant vector. The gene was induced to express fusion protein in E. coli host strain BL21(DE3) and the fusion protein was identified by Western blot. BALB/c mice were immunized with purified protein to test its immunogenicity. Then the purified protein was used as antigen to test the serum of Mp infected patient by ELISA, and compared with the Mp whole cell antigen. Results The P1-534 protein was successfully expressed and purified. ELISA data showed that P1-534 protein could elicit high levels of IgG in immunized mice, the sensitivity and specificity of P1-534 were determined to be 85.00% and 97.67%, while the Mp whole cell antigen were 72.50% and 74.42%. Conclusion The results conformed that the recombinant epitope has certain immunogenicity,and its sensitivity and specificity are better than Mp whole cell antigen. P1-534 protein can be used as an antigen for immunodiagnosis of Mp infection.

Objective To compare the capabilities of culture method, polymerase chain reaction ( PCR) and serological test in identifying Mycoplasma pneumoniae infection in children with confirmed com-munity acquired pneumonia. Methods Bronchoalveolar lavage fluid and serum samples were collected from hospitalized children with community acquired pneumonia in Capital Institute of Pediatrics from March to May in 2016. Three methods, traditional culture method, PCR and serological test, were respectively used to de-tect Mycoplasma pneumoniae infection in those children. Statistical analysis was performed by using SPSS18. 0 software and chi-square test. Results Seventy-nine children with community acquired pneumonia were enrolled in this study. Eight (10. 13%) patients were diagnosed with Mycoplasma pneumoniae infec-tions by the traditional culture method with an average positive culture period of 21 days. Twenty-three (29. 11%) patients showed positive results by using PCR analysis, including the 8 patients identified by the culture method. Forty-one (51. 90%) patients were found to be positive for Mycoplasma pneumoniae infec-tions by the serological test. However, four negative samples identified by the serological test were confirmed to be positive by PCR analysis, including two positive samples confirmed by the culture method. Statistical analysis showed that the differences in positive rates detected by using the three methods were statistically significant. Conclusion It is recommended that both serological test and PCR analysis should be used in combination with clinical symptoms for a comprehensive assessment of Mycoplasma pneumonia infection in children.

Objective To inspect the source of an outbreak with Mycoplasma pneumoniae (Mp).Methods We carried out real-time PCR to analyze specimens collected from pediatric patients in Beijing during January 2010 to May 2012,diagnosed as pneumonia or a respiratory infection according to clinical symptoms.These positive samples were analyzed by the M-P typing system(M:multiple-locus variable-number tandem-repeat analysis,MLVA; P:P1-restriction fragment length polymorphism analysis,P1-RFLP).Results Sixty-nine specimens were tested positive to Mp by the real-time PCR in 446 specimens from pediatric patients.The infection rate was 11.69％,15.56％ and 20.00％ respectively in 2010,2011 and the first half of 2012.According to the M-P system,11 distinct genotypes were identified from 69 positive specimens,M43562P1 and M53562P1 were the two main genotypes that showed an increasing trend from 2010 to 2011,and M33562P1 and M63562P1 showed an increasing trend from 2011 to 2012 in China.Conclusion During this international Mp epidemic,the infection rate of Mp was also increase in Beijing in 2011,and M43562P1 and M53562P1 were the two main genotypes.Among them,M43562 were consistent with pop genotypes in Europe,and M53562 were consistent with pop genotype in Israel.The M-P system would be valuable to monitor the epidemic of Mp in different countries in the world.

Objective:To evaluate the rapid nucleic acid amplification detection of Mycoplasma pneumoniae (MP)-DNA and MP-RNA in the diagnosis of MP infection and therapeutic values in children. Methods:Patients who were diagnosed with pneumonia were enrolled from the Department of Respiration, Children′s Hospital of Capital Institute of Pediatrics from January 2018 to December 2018.Specimens were detected using the MP and Macrolide-Resistant isolates Diagnostic Kit (PCR Fluorescence Probing, Jiangsu Mole Bioscience Co., Ltd.) and MP Diagnostic Kit (Isothermal RNA amplification, Shanghai Rendu Biotechnology Co., Ltd.).Results:Among them, 42.1%(840 cases) of the 1 994 cases were positive for MP-DNA, and the macrolide associated gene mutations were detected in 96.0% (806/840 cases) of them, while 33.9% (551 cases) of 1 624 cases were positive for MP-RNA.Seven hundred and fifty-eight specimens were simultaneously detected by adopting MP-DNA and MP-RNA, and the positive rate was 43.1% (327/758 cases) and 36.7% (278/758 cases), accordingly, which were inconsistent (Kappa=0.604) in 613 (80.9%, 613/758 cases) cases, with significant differences ( χ2=6.60, P=0.01). Part of the specimens were rechecked with the interval of 7 days: MP-RNA was negative in 70.1% (47/67 cases) specimens and MP-DNA was negative in 36.1% (22/91 cases) specimens ( χ2=33.20, P<0.01). Conclusions:The positive detection rate of MP was at a high level in 2018, in Beijing, China.The results of MP-DNA and MP-RNA are consistant.But RNA detection can help to diagnose MP in the early stage, and monitor the survival of MP and its efficiency.

BACKGROUND:The incidence of low serum level of vitamin D in patients undergoing hip arthroplasty and its impact has not been reported in China, indicating that it has not been brought to the forefront. OBJECTIVE:To determine the prevalence of low serum level of vitamin D in patients before total hip arthroplasty and its relationship with the hip function scores. METHODS:Forty-eight hips from 48 patients undergoing primary hip arthroplasty from July 2013 to August 2014 in the First Affiliated Hospital of Suzhou University were enrol ed. According to the serum level of vitamin D, patients were assigned to low-level (<20μg/L) and high-level (20≥μg/L) groups. The general information of patients, the hip function scores before and after replacement at the last fol ow-up in the two groups were observed and compared. The relationship between the serum level of vitamin D and the hip function scores before and after replacement was analyzed by multiple linear regression analysis. And the average fol ow-up was 12 months (11-14 months). RESULTS AND CONCLUSION:(1) The incidence of low vitamin D level was 82%(20 ng/mL serving as standard). (2) Compared with patients with high vitamin D level, patients with low level of vitamin D had lower preoperative Harris scores and Merle D′Aubigne-Postel score (P<0.05), and also at the last fol ow-up (P<0.05. (3) Based on the preoperative and postoperative Harris, the multiple linear regression analysis showed that there was a positive correlation between the level of vitamin D and Harris score both preoperatively and postoperatively (P<0.05). (4) These results suggest that there is a higher incidence of low level of vitamin D in patients undergoing arthroplasty, and hip function scores before and after replacement in patients with low level of vitamin D are lower than the high level patients. Moreover, there is a positive correlation between the level of vitamin D and the hip joint function scores. Therefore, it is advisable to supplement vitamin D and calcium preoperatively, and the level of vitamin D wil be helpful for disease assessment and prognosis.

Objective To evaluate the efficiency of using nested PCR in restriction fragment length polymorphism analysis (P1-RFLP) for genotyping Mycoplasma pneumonia (M.pneumonia) in clinical specimens.Methods Based on the gene sequence of RepMp4 and RepMp2/3 in P1 gene of reference strains M129 (type 1) and FH (type 2),two sets of inner primers were designed with a HaeⅢ restriction enzyme site (GGCC).The nested PCR was set up to detect the target DNA in clinical specimens.The amplification products were mixed and digested with Hae Ⅲ enzyme.The genotypes were analyzed by comparing with various restriction maps and the results were verified by sequencing analysis.The concentration of DNA extracted from standard and clinical strains were detected by ten-fold dilution to evaluate the sensitivity of nested PCR-P1-RFLP and P1-RFLP.M.pneumonia-positive specimens isolated from Beijing in 2012 were analyzed by the nested PCR-P1-RFLP and the results were compared with those by P1-RFLP analysis.Results The nested PCR-P1-RFLP could effectively genotype M.pneumonia in clinical specimens and the results were consistent with those by sequencing analysis.The sensitivity of new assay was 103 times higher than that of the original P1-RFLP.Of the 115 M.pneumoniae positive clinical specimens,97.4％ (112/115) were type 1 and the rest were type 2.Conclusion The nested PCR-P1-RFLP shows high efficiency for genotyping of M.pneumonia in clinical specimens.It might be useful for the surveillance of M.pneumoniae infection.

We analyzed genetic evolution characteristics of avian influenza A (H7N9) virus isolated in Zhaoqing,China,2014-2016.Nucleic acid were extracted and sequenced from 17 samples of H7N9 positive cases in Zhaoqing.Genetic characteristics of homology and important amino acid sites were analyzed by using BioEdit5.0 and MEGA6.0.The evolutionary trees were constructed by Neighbor-Joining and the referenced sequences were downloaded from GenBank,Eight nucleic acid fragments from 7 strains of H7N9 viruses were successfully generated.The highest homology was found in HA gene with A/chicken/Dongguan/695/2014(H7N9),and NA gene with A/chicken/Dongguan/1075/2014(H7N9).The internal genes were high homology with avian H7N9 and H9N2 virus from Dongguan and Shenzhen in Guangdong,China.The HA and NA genes were directly evolved in the Pearl River Delta evolution branch with the H7N9 sequences from the cities of Dongguan,Guangzhou and Shenzhen,while the sequences from the provinces of Anhui,Zhejiang,and Jiangsu were in the Yangtze River Delta evolution branch.There were 2 alkaline amino acids in cleavage site of HA,2 mutations (G186V and Q226L) in the crucial sites related with the receptor of HA protein,1 mutation (E627K) in PB2 protein,and 1 drug resistance mutation (S31N) in M2 protein.And no evidence of neuraminidase resistance in NA protein was found.In conclusion,the H7N9 virus for human infection in Zhaoqing may originate from avian H7N9 and H9N2 viruses,which circulated in the Pearl River Delta region of Guangdong from 2013 to 2014.The mutations of G186V,Q226L and E627 K might be related with high susceptibility to human beings.

Objective To investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes among clinical isolates of Klebsiella pneumoniae (K.pneumoniae) in pediatrics.MethodsA total of 131 non-duplicate clinical isolates of K.pneumoniae were collected in the Affiliated Children′s Hospital of Capital Institute of Pediatrics from 2010 to 2012.PMQR genes [qnrA, qnrB, qnrS, aac(6′)-Ⅰb-cr and qepA], mutations in the quinolone resistance-determining region (QRDR) and extended spectrum β-lactamases (ESBLs) genes in those strains were analyzed by PCR.Minimum inhibitory concentrations (MIC) of different antibiotics against those K.pneumoniae strains were determined by broth microdilution method and E-test according to the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI).Transferability of the PMQR genes was examined by conjugation test with the sodiumazide-resistant Escherichia coli J53.Results Among the 131 isolates, 9.92% were resistant to quinolone and 30.5% were positive for PMQR genes, including 6.87% harboring qnrB gene, 22.9% harboring qnrS gene and 4.58% harboring aac(6′)-Ⅰb-cr gene.Neither qnrA-positive nor qepA-positive strain was detected.Among these PMQR genes-positive isolates, 90% were ESBLs-producing strains and two presented mutations in gyrA and parC genes.Conjugation test showed that these PMQR genes could be transferred horizontally and the ciprofloxacin resistance increased 2 to 32 folds in transconjugants.Conclusion This study indicates that the PMQR gene-carrying rate is high in K.pneumoniae strains isolated in paediatrics in China.Most of the PMQR gene-positive strains are also ESBLs-producing strains.The PMQR genes could be transferred horizontally in bacteria.